Mass spectrometry (MS) has been the driving force behind the development of proteomics and has had a large impact in the fields of molecular and cellular biology. Concurrently, there has been growing interest in using native mass spectrometry to investigate protein complexes and other assemblies with masses into the MDa range. However, there are challenges associated with the mass analysis of such large objects. The main issue is that the peaks in the m/z spectrum broaden and shift due to mass heterogeneity, either intrinsic or due to complex formation. Poorly resolved peaks in the m/z spectrum prevent charge state assignment and subsequent mass deduction. In particular, viruses have a proclivity for being heterogeneous in mass because they have the ability to encapsidate varying amounts of genetic material. Earlier studies demonstrated the feasibility of using time-of-flight mass spectrometry to measure the m/z spectrum of the ˜2.5 MDa bacteriophage MS2 capsid, albeit without sufficient charge state resolution to calculate an accurate mass of the complex. More recently, high resolution m/z spectra of empty hepatitis B virus (HBV) capsids assembled from truncated proteins lacking the C-terminal RNA-binding domain have been reported. However, the m/z spectrum for HBV assembled from the full-length capsid protein lacked charge state resolution due to heterogeneity.
AAV vectors have emerged at the forefront of gene therapy due to their lack of pathogenicity, relatively low immunogenicity and persistent gene expression in different tissue types. From a structural perspective, this helper-dependent parvovirus has a non-enveloped, icosahedral capsid ˜25 nm in diameter that packages a single-stranded DNA (ssDNA) genome ˜4.7 kb in length. Despite promising outcomes in several clinical trials, a recurring concern noted in hemophilia gene therapy clinical trials is the potential for vector dose-related immunotoxicity in patients. Although resolvable by administration of anti-inflammatory steroids such as methyl prednisolone, several studies have indicated that the composition of clinical AAV vector preparations can influence these outcomes. In this regard, recombinant AAV vector preparations can contain different levels of full or partial genome-containing particles as well as empty virions. Such particle diversity can be attributed to multiple factors such as genome packaging efficiency, production methods, downstream purification techniques and storage conditions.
Though AAV packages ssDNA, the use of a self-complementary (sc) DNA genome bypasses the rate-limiting second-strand synthesis process and leads to more efficient and rapid onset of trans gene expression. scDNA is a double-stranded DNA molecule formed by intramolecular base paring of two single-stranded vectors joined by a hairpin. Because scDNA is packaged as a single strand, the total length of the DNA is limited to approximately 4.7 kb so that the effective length of the unique transgene sequence is halved. Upon release into the host cell, scDNA anneals into the base-paired form. Though scAAV vectors show promise in the clinic, their characterization remains a challenge.
Currently, electron microcopy (EM) is utilized to characterize the ultrastructural composition of AAV vector preparations. Although useful, this method is time consuming, subjective, and relies on large datasets to obtain an accurate representation of AAV particle diversity. While this technique can distinguish empty virions from genome-containing particles, EM may not help resolve partial or truncated genome-containing particles and free vector genomic DNA. Also, current quantitative PCR-based methods, cannot help distinguish between partial/truncated vector genomes from fully packaged genomes. Recently, Burnham et al demonstrated the use of analytical ultracentrifugation as a low-resolution technique for the characterization of recombinant AAV vectors. Thus, the development of cutting edge methods that can help analyze ultrastructural heterogeneity in recombinant AAV vector preparations at high resolution is an unmet need in the gene therapy field.
The present invention addresses a need in the art for protocols that allow for resolution of different components of a preparation of virus particles.